ISA_V paper

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EFSA Journal 2012;10(11):2971

Suggested citation: EFSA Panel on Animal Health and Welfare (AHAW); Scientific Opinion on infectious salmon anaemia.EFSA Journal 2012;10(11):2971.[22 pp.] doi:10.2903/j.efsa.2012.2971. Available online: www.efsa.europa.eu/efsajournal

European Food Safety Authority, 2012

SCIENTIFIC OPINION

Scientific Opinion on infectious salmon anaemia (ISA)1

EFSA Panel on Animal Health and Welfare (AHAW)2,3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT

Atlantic salmon is the only species in which the disease infectious salmon anaemia (ISA) has beenobserved naturally. Initial reports of findings of infectious salmon anaemia virus (ISAV) before 2002,did not distinguish between non virulent HPR0 and virulent HPRviruses, thus making interpretationof older findings difficult in the light of current knowledge. Following a request from the EuropeanCommission, EFSA was asked to deliver a scientific opinion on the relationship between HPR0 andHPR, the risk of HPR ISAV emerging from HPR0 ISAV, and possible risk factors for such anemergence. HPR0 ISAV does not cause clinical disease in Atlantic salmon; however, it causes atransient subclinical infection and replicates mainly in gills. There is no evidence for HPR0 ISAVleading to natural infection and replication in fish species other than Atlantic salmon. Virulent ISAVhave deletions in the HPR region of the HE gene and they have either an insertion or the Q266Lmutation in the F gene. The most plausible hypothesis is that virulent ISAV (HPR) is derived fromHPR0 ISAV. This is further supported by the close association between the genetic relatedness andspatio-temporal distances of virus strains in solitary outbreaks. Epidemiological and historical datafrom solitary disease outbreaks indicates that the risk of HPR ISAV emerging from HPR0 is low,

but not negligible. The risk factors for HPR emergence from HPR0 are unknown. Nevertheless, anyfactor that affects virus replication or host susceptibility could possibly influence the risk ofemergence. More research is needed on the drivers for transition from HPR0 to HPR and factorsaffecting host susceptibility and thereby emergence of clinical disease. A quantitative assessment ofthe different evolutionary forces for ISA would be useful, as well as the prevalence of ISAV HPR0 in

farmed and wild Atlantic salmon.

European Food Safety Authority, 2012

KEY WORDS

infectious salmon anaemia virus, isavirus, virulence, highly polymorphic region, HPR0

1 On request from the European Commission, Question No EFSA-Q-2012-00060, adopted on 16 November 2012.2 Panel members: Edit Authie, Charlotte Berg, Anette Btner, Howard Browman, Ilaria Capua, Aline De Koijer, Klaus

Depner, Mariano Domingo, Sandra Edwards, Christine Fourichon, Frank Koenen, Simon More, Mohan Raj, LiisaSihvonen, Hans Spoolder, Jan Arend Stegeman, Hans-Hermann Thulke, Antonio Velarde, Ivar Vgsholm, Preben

Willeberg and Stphan Zientara. Correspondence: [email protected] Acknowledgement: The Panel wishes to thank the members of the Working Group on infectious salmon anaemia: Edgar

Brun, Debes Christiansen, Philippe Lemey, Niels Jrgen Olesen, Rob Raynard, Espen Rimstad, Fulvio Salati, Mike Sharp(chair until July 2012), Liisa Sihvonen and Ivar Vgsholm (chair from July 2012) for the preparatory work on this

scientific opinion and EFSA staff, Per Have for the support provided to this scientific opinion.


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EFSA Journal 2012;10(11):2971 2

SUMMARY

Following a request from the European Commission, the EFSA Panel on Animal Health and Welfarewas asked to deliver a scientific opinion on infectious salmon anaemia (ISA). ISA is a fish disease

caused by an orthomyxovirus (infectious salmon anaemia virus, ISAV) affecting Atlantic salmon,which has been responsible for high mortalities in salmon producing countries since its first detection

in Norway in the mid-1980s.

This opinion reviews the available scientific evidence on the relationship between HPR0 and HPR,and addresses the risk of HPR ISAV emerging from HPR0 ISAV and, if relevant, indicates the risk

factors for such an emergence.

Pathogenic HPR ISAV variants cause a systemic infection, infecting endothelial cells of the blood

circulatory system whereas HPR0 ISAV does not cause clinical disease in Atlantic salmon but causesa transient subclinical infection and replicates mainly in gills.

ISAV can be genetically differentiated on the basis of the sequence of a highly polymorphic region(HPR) of genomic segment 6 which encodes the Haemagglutinin-Esterase (HE) protein. A deletion

within the HPR region (HPR ISAV) is necessary for pathogenicity. ISAV without any deletions inthe HPR region (Hereinafter: HPR0 ISAV) has been reported only in apparently healthy fish and hasnever been associated with clinical ISA disease.

HPR0 has been detected in farmed Atlantic salmon from several countries and it has also beendetected in wild Atlantic salmon in the Faroe Islands and Norway. There is currently no evidenceindicating that HPR0 ISAV naturally infects and replicates in species other than Atlantic salmon. The

finding of HPR0 ISAV in wild Atlantic salmon indicates that a reservoir outside the farmed salmonpopulation may exist.

ISAV isolates vary in virulence, as observed by differences in disease development and clinical signsin field outbreaks as well as in experimental trials. All ISAV isolates from ISA disease outbreaks havedeletions in the HPR region with respect to the HPR0 variant. In addition, all virulent strains of ISAVhave either an amino acid substitution or a short amino acid insertion immediately upstream ordownstream of the putative arginine cleavage site in the fusion (F) protein.

The hypothesis that virulent HPR ISAV is derived from HPR0 ISAV by deletions in the HPR of theHE molecule provides the best fit with current knowledge and epidemiological evidence.Epidemiological and historical data from solitary disease outbreaks indicate that the risk of emergenceof virulent ISAV is low but not negligible.

Generic biosecurity measures such as segregation of generations, caution regarding contact points

(water, equipment), sanitary handling of dead fish, cleaning and disinfection, and synchronous

fallowing appear to have a good effect in terms of prevention and control of ISA.

Prior adaptive immunity provides some protection against subsequent infection with virulent ISAV.Little is known about antigenic variation in the haemagglutinin-esterase gene of ISAV and it is notpossible to conclude whether this may impact population immunity. Likewise, it is not known if or to

what extent prior infection with HPR0 ISAV may induce some degree of protective immunity.

The evolutionary relationship between virulent and low-virulent ISAV forms, where HPR0 mutatesinto a virulent form of the ISAV, appears plausible. However, no predisposing risk factors have beendemonstrated or suggested to drive or increase such an evolutionary process.

Based on general virological knowledge, the risk of emergence of HPR and subsequent development

of disease can be expected to be related to the overall replication of HPR0 ISAV and the presence ofsusceptible hosts. Any factor that affects replication or host susceptibility would, therefore, also

influence the risk of emergence of HPR ISAV.


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TABLE OF CONTENTS

Abstract .................................................................................................................................................... 1Summary .................................................................................................................................................. 2Table of contents ...................................................................................................................................... 3Background as provided by the European Commission ........................................................................... 4Terms of reference as provided by the European Commission ................................................................ 4Assessment ............................................................................................................................................... 51. Introduction ..................................................................................................................................... 5

1.1. The disease .............................................................................................................................. 51.2. History of ISAV diagnosis ...................................................................................................... 51.3. Species range .......................................................................................................................... 51.4. ISAV infection ........................................................................................................................ 6

2. The capability of HPR0 ISAV to cause clinical disease (ToR1) ..................................................... 62.1. Clinical signs ........................................................................................................................... 72.2. Pathogenesis ............................................................................................................................ 72.3. Immune responses ................................................................................................................... 72.4. Geographical distribution of HPR0 ISAV .............................................................................. 8

3. What is the risk of HPR ISAV emerging from HPR0 ISAV (TOR 2a) ........................................ 83.1. Genome of ISAV .................................................................................................................... 83.2. Virulence of ISAV .................................................................................................................. 93.3. Phylogenetics and evolutionary dynamics of ISAV ............................................................... 9

4. Relevant risk factors for emergence of HPR (ToR 2b)............................................................... 114.1. ISAV transmission and spread .............................................................................................. 114.2. HPRdelta emergence from endemic HPR0 infection ............................................................ 12

Conclusions and recommendations ........................................................................................................ 12References .............................................................................................................................................. 13Appendix ................................................................................................................................................ 19A. Diagnosis of ISA ........................................................................................................................... 19

Propagation of ISAV in cell lines ...................................................................................................... 20

Direct methods ................................................................................................................................... 20Detection of ISAV (HPR) by RT-PCR (interpretation for surveillance and reservoirs) ............. 20

Glossary and abbreviations .................................................................................................................... 22


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BACKGROUND AS PROVIDED BY THEEUROPEAN COMMISSION

ISA is a fish disease listed in Part II of Annex IV to Council Directive 2006/88/EC on animal healthrequirements for aquaculture animals and products thereof, and on the prevention and control of

certain diseases in aquatic animals.

ISA mainly affects Atlantic salmon and since its first detection in Norway in mid-1980s has beencausing great mortalities in salmon producing countries (Canada, Chile, USA, Norway, Faroe Islands).Within the EU, there have been only two outbreaks, in Scotland, UK (1998eradicated, 2009undereradication4).

Variants of ISAV have been genetically differentiated on the basis of the sequence of a highlypolymorphic region (HPR of genomic segment 6 which encodes the Haemagglutinin-Esterase (HE)

protein). A deletion within the HPR region (hereinafter: HPR ISAV) in certain ISAV variantsappears to be a consistent indicator of pathogenicity. ISAV without any deletions in the HPR region(Hereinafter: HPR0 ISAV) has been reported only in apparently healthy fish and to our knowledge hasnever been associated with clinical ISA disease.

The current prescribed diagnostic methods for confirmation of ISA in the OIE Manual of DiagnosticTests for Aquatic Animals (2009) and Commission Decision 2003/466/EC establishing criteria forzoning and official surveillance following suspicion or confirmation of the presence of infectious

salmon anaemia (ISA) do not allow for the detection of HPR0 ISA.

However, existing reverse-transcription polymerase chain reaction (RT-PCR) diagnosticmethodologies do allow the detection and differentiation of both HPR0 and HPR ISAV variants.

Furthermore, amplification of the segment 6 and subsequent sequencing is an accurate method fordiscrimination between HPR0 and HPR ISAV variants.

HPR0 ISA appears to be widely spread throughout the world, both in areas which are infected withand free from HPR ISA.

The Commission is evaluating whether it is appropriate, proportionate and necessary to apply risk

management measures to HPR0 ISA. To facilitate that evaluation it is appropriate and necessary toassess the risks posed by HPR0 ISA for the health of aquatic animals, in particular Atlantic salmon.

TERMS OF REFERENCE AS PROVIDED BY THEEUROPEAN COMMISSION

In view of the above, and in accordance with Article 29 of Regulation (EC) 178/2002, the Commissionrequests EFSA for a scientific opinion on the HPR0 variant of Infectious salmon anaemia (HPR0ISA), and in particular to assess;

1. The capability of HPR0 ISA to cause clinical disease.

2. The risk of HPR-deleted ISA emerging from HPR0 ISA and, if relevant, indicating factors forsuch an emergence.

4Declaration of freedom submitted to SCoFCAH on March 9, 2012.


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ASSESSMENT

1. Introduction1.1. The diseaseInfectious salmon anaemia (ISA) is a fish disease caused by infectious salmon anaemia virus (ISAV),an orthomyxovirus, affecting Atlantic salmon (Salmo salar) by inducing a systemic and lethalcondition characterised by severe anaemia and variable haemorrhages and necrosis in several organs(Rimstad et al., 2011a). Current diagnostic procedures are described in Annex A.

So far the disease ISA has been described only in farmed salmon, and the Atlantic salmon is the onlyspecies in which the disease has been observed naturally. Since its first detection in Norway in themid-1980s, ISA has been responsible for high mortalities in salmon-producing countries with higheconomic losses. The outbreak in Scotland in 1998 was eradicated at a cost of over 20 million(Hastings et al., 1999); the epidemic in Chile cost considerably more (Asche et al., 2009) and wassufficient to lead to shortage in supply to the market, which affected global prices of salmon. ISA is

now a global disease, and outbreaks have been recorded in most countries with a significant Atlanticsalmon aquaculture industry (Table 1).

Table 1: Major outbreaks of infectious salmon anaemia

Year Country Reference

1984 Norway Thorud and Djupvik, 1988

1996 Canada Mullins et al., 1998

1998 Scotland, UK Rodger et al., 1998

2000 Faroe Islands Christiansen et al., 2011

2001 USA Bouchard et al., 2001

2007 Chile Godoy et al., 2008

Mardones et al., 2009

2009 Scotland, UK Murray et al., 2010

1.2. History of ISAV diagnosisThe initial reports of findings of ISAV did not distinguish between HPR0 and HPR, makinginterpretation of older studies more difficult in the light of current knowledge. The distinction betweenHPR0 and HPR ISAV was established in 2002 (Mjaaland et al., 2002a; Nylund et al., 2003). Basedon current evidence and subsequent sequencing of associated ISAV isolates, most of these olderstudies probably reflect infections with HPR ISAV.

1.3. Species rangeA susceptible species is defined by Council Directive 2006/88/EC as any species in which infection by

a disease agent has been demonstrated in natural cases or by experimental infection that mimics thenatural pathways. This definition was used for the EFSA opinion Aquatic species susceptible todiseases listed in Directive 2006/88/EC (EFSA, 2008) and also informed the current opinion.

Subclinically ISAV-infected feral Atlantic salmon, brown trout and sea trout (S. trutta) and escaped,farmed rainbow trout (Oncorhynchus mykiss) have been identified by RT-PCR (Raynard et al., 2001;Plarre et al., 2005). Waterborne transmission in juvenile rainbow trout, including high mortalities in

genetically susceptible fish strains, have been found in some experimental set-ups (Biacchesi et al.,2007), while serial passage of a virulent strain of ISAV in larger rainbow trout did not produce clinical

disease (Olsen et al., 2012). Positive RT-PCR for ISAV has also been reported in wild Atlantic cod(Gadus morhua), alewife (Alosa pseudoharengus) and pollock (Pollachius virens) (MacLean et al.,2003; Rolland, 2004), but contamination by virus present in the surrounding water cannot be excludedas the fish were collected close to cages with Atlantic salmon exhibiting ISA. In other studies, notransmission in cohabitant challenge of Atlantic cod has been found (Snow and Raynard, 2005; Grove


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et al., 2007), and pollock cohabitating with farmed Atlantic salmon in sea cages remained PCRnegative when harvested together with salmon experiencing increased mortality due to ISA (McClure

et al., 2004) and was also found negative for ISAV following exposure by intraperitoneal injection ofvirus or by cohabitation with ISAV-infected Atlantic salmon (Snow et al., 2002). Based on these

findings, both Atlantic salmon,brown trout and sea trout are candidates as natural hosts for HPR0

ISAV. There are no indications that ISAV can infect blue mussel (Mytilus edulis) or scallops (Pectenmaximus) or that these shellfish play any role as reservoirs for ISAV (Skr and Mortensen, 2007).

Atlantic salmon is the only species in which the disease ISA has been repeatedly observed naturally.There is a report of isolation of ISAV from Coho salmon (O. kisutch) with jaundice disease in Chile

(Kibenge et al., 2001a), but this has not been reported elsewhere.

1.4. ISAV infectionThe development of more sensitive methods for virus detection by PCR during the 1990s (Mjaaland etal., 1997) enabled studies providing evidence of ISA virus infection in apparently healthy wild fish(feral Atlantic salmon, brown trout and sea trout, and escaped, farmed rainbow trout) (Raynard et al.,2001; Plarre et al., 2005). Refinements to molecular methods enabled the description of genomic

sequences of ISA virus in wild salmonids which were hypothesised to show a full-length sequence ofthe HPR of the haemagglutinin-esterase (HE) gene (Mjaaland et al., 2002b). Thus, the hypothesis thatdeletions of HPR0 were required for emergence of HPR variants (HPR) associated with virulentforms of ISAV was derived (Mjaaland, Hungnes, et al., 2002; Nylund et al., 2003).

ISAV can be genetically differentiated on the basis of the sequence of the HPR of genomic segment 6which encodes the HE protein. Deletions within the HPR region (HPR ISAV) have been identified inall virulent isolates causing clinical ISA disease and appear to be necessary for pathogenicity.

HPR0 ISAV appears to be widely distributed, both in areas infected with and areas free from HPRISAV and clinical disease. From a disease control point of view it is important to understand the

dynamics and interrelationship between HPR0 and HPR ISAV and, more particularly, the likelihoodof and the reasons why HPR arises from a background source of HPR0 ISAV .

In view of the above, the Commission is evaluating whether it is appropriate, proportionate andnecessary to apply risk management measures to HPR0 ISA, in addition to those applied to HPRISAV. EFSA has been asked for a scientific opinion on the HPR0 variant of ISAV and to assess therisks posed by HPR0 ISA for the health of aquatic animals, in particular Atlantic salmon. The terms ofreference (ToRs) provided by the Commission can be formulated as three questions:

1. Can HPR0 ISA cause clinical disease?2. What is the risk of HPR-deleted ISA emerging from HPR0 ISA and, if relevant, indicating

factors for such an emergence

a. What is the risk of HPR-deleted (HPR) ISAV emerging from HPR0 ISAV?b. What are the factors relevant for such an emergence?

The above questions will be addressed on the basis of published literature, other evidence and theexpertise of the scientists engaged in the opinion from the panel and Working Group.

2. The capability of HPR0 ISAV to cause clinical disease (ToR1)ISA is a systemic and lethal condition and clinical signs suggest circulatory failure. So far only HPR

ISAV variants have been reported to cause disease in Atlantic salmon.


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2.1. Clinical signsThe classic external signs comprise pale gills, exophthalmia, distended abdomen, blood in the anterioreye chamber and sometimes skin haemorrhages, especially of the abdomen, as well as scale pocketoedema. Affected fish are lethargic, keeping close to the wall of the net pen. The position of the fish inwater is often vertical and some diseased fish show a corkscrewing swimming behaviour. The

nutritional status is usually normal, but diseased fish have no feed in the digestive tract. Thedevelopment of the disease may be insidious with low mortality and few clinical signs for months;however, cumulative mortality can reach very high levels (up to 90%).

At necropsy, a variable set of haemorrhages and necroses in several organs are usually observed.Typical findings also include severe anaemia, ascites and congestion and enlargement of the liver andspleen (Evensen et al., 1991).

2.2. PathogenesisISA can be experimentally reproduced in Atlantic salmon by intra-peritoneal injection with HPRISAV or through infected cohabitants. The incubation period in experimental infection is usually 10

20 days (Rimstad and Mjaaland, 2002). The most likely route of virus entry following cohabitationwith infected fish is the gills, but other ports of entry cannot be excluded (Mikalsen et al., 2001). In

infection experiments, virulent ISAV rapidly produces a systemic infection and has been detectedthroughout the body 5-10 days post infection with a peak in viral load at approximately 15 days postinfection (Totland et al., 1996; Rimstad et al., 1999).

The major target cells for virulent ISAV in vivo are endothelial cells (Hovland et al., 1994; Koren andNylund, 1997; Falk et al., 1998; Gregory, 2002). ISAV specifically binds 4-O-acetylated sialic acid at

the endothelial cell surface, and the esterase part of the HE hydrolyses ISAV cell surface receptors(Hellebo et al., 2004). ISAV infects cells via the endocytic pathway and the fusion between virus andcell membrane takes place in endosomes (Eliassen et al., 2000). Virus-infected endothelial cells can befound in any organ (Aamelfot et al., 2012), leading to circulatory collapse, often accompanied by

extreme anaemia, haemorrhages and necrosis of internal organs (Hovland et al., 1994).

No experimental infection has been carried out so far with HPR0 ISAV. However, this variant has alsobeen detected in naturally infected salmon, most often in the gills (McBeath et al., 2009; Christiansenet al., 2011). As opposed to the systemic and severe disease caused by HPR ISAV, HPR0 ISAVreplicates in (D.H. Christiansen, personal communication) and causes a localized infection of salmongills with no signs of disease and only occasional spread to other organs (Christiansen et al., 2011).

Parallel testing of kidney, heart and gill tissue for the presence of HPR0 ISAV by real-time RT-PCRdisclosed a significantly higher overall detection in gill tissue compared with kidney and heart. Also,the load of HPR0 ISAV virus in positive gills was generally much higher than in kidneys and hearts(Christiansen et al., 2011; Lyngstad et al., 2011). Thus, HPR ISAV and HPR0 ISAV show differentinfection patterns and tissue tropism, a pattern similar to that found in wild aquatic birds in which low-

pathogenic avian influenza virus causes a subclinical, transient, mucosal infection whereas highlypathogenic influenza causes a systemic and lethal infection in poultry.

2.3. Immune responsesBoth cellular and humoral immune responses against ISAV have been demonstrated in Atlanticsalmon (Falk and Dannevig, 1995; Kibenge et al., 2002; Mjaaland et al., 2005; Lauscher et al., 2011).

Studies by western blotting of the Atlantic salmon antibody response to ISAV revealed that ISAVantibodies bound exclusively to the viral nucleoprotein and hemagglutinin protein (Clouthier et al.,2002; Rimstad, Dale, et al., 2011). Antibody-mediated enhancement of virus infection may beinvolved in the pathogenesis of ISA because ISAV targets leucocytic cells, and Fc receptors for fishimmunoglobulin M have been demonstrated on fish leucocytes.

Prior adaptive immunity conferred by inactivated field and experimental vaccines provides someprotection against subsequent infection with virulent ISAV (Jones et al., 1999; Lauscher et al., 2011).


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DNA vaccines containing the HE gene can also confer protective immunity (Mikalsen et al., 2005),indicating that this surface glycoprotein is antigenic and involved in stimulating a protective immune

response. Little is known about antigenic variability in the HE protein of ISAV and it is therefore notpossible to conclude whether this may impact population immunity as is seen with influenza A

viruses. Likewise, it is not known if cross protection may be achieved against different variants and to

what extent prior infection with HPR0 ISAV may induce some degree of protective immunity toHPR variants.

2.4. Geographical distribution of HPR0 ISAVThe first detection of HPR0 ISAV was done on gill tissue from a wild-caught Atlantic salmon inScotland (Cunningham et al., 2002). In addition to Scotland, HPR0 ISAV has also been detected infarmed Atlantic salmon from the Faroe Islands, Norway, Canada, Chile and Denmark (N.J. Olesen,personal communication).

HPR0 has also been detected in wild Atlantic salmon in the Faroes and Norway. Three out of 88confirmed wild Atlantic salmon caught by a Faroese research vessel at the feeding grounds in theNorth Atlantic were HPR0 positive (D.H. Christiansen, personal communication). Furthermore, 4 out

of 305 Atlantic salmon caught in rivers in mid-Norway were found to be positive by PCR. Viral RNAfrom one of them was sequenced to HPR0, clustering phylogenetically with the Faroes cluster. Theamount of RNA from the other three was too scarce for sequencing, but still empirically indicated thepresence of HPR0. All salmon were caught in an area with on-going ISA outbreaks with virussubtypes associating with a cluster different from that identified as HPR0. The four positive ones wereall from the same river and confirmed as wild salmon according to fish scale examination. (R.Grntvedt and T. Lyngstad, personal communication).

The Faroe Islands documented findings of HPR0 in Atlantic salmon in their monitoring from 2005 to2009. HPR0 was detected on gills 113 months post sea transfer (mean 7.7 months). The various

cohorts (49) were sampled 512 times each year, and the presence of HPR0 on gills showed peakedtransient infection profile with peak prevalence up to 100 % lasting for 4 months. Almost all of thecohorts were positive for HPR0. No clinical disease or histopathological consequences have beenreported in association with this HPR0 infection in the Faroes (Christiansen et al., 2011).

In Chile, all ISAV strains detected in 2011 were identified as HPR0 (Kibenge et al., 2012). No

outbreaks were observed and HPR was not detected.

In a retrospective study in Norway (Lyngstad et al., 2012), ISAV was present in 23 % of 210 cohortsof marine farmed Atlantic salmon along the coast, with no suspicion of ISA. HPR0 ISAV wasconfirmed in 59 % of these ISAV-positive groups. The rest of the positive groups were not sequenceddue to lack of RNA, but the low titres may indicate the presence of HPR0.

The groups were sampled once and at various points in time after sea transfer. In other screeningstudies, HPR0 has been detected in gill samples from juvenile salmon and in brood stock in thefreshwater environment (M. Devold and D. H. Christiansen, personal communication). A low level ofHPR0 has also been detected in ovarian fluid of farmed Atlantic salmon (D. H. Christiansen, personalcommunication).

3. What is the risk of HPRISAV emerging from HPR0 ISAV (TOR 2a)In order to address this question the genomic structure of ISAV is briefly described and the evidenceavailable regarding virulence factors and phylogenetics and evolutionary dynamics is considered.

3.1. Genome of ISAVThe ISA virus belongs to the genus Isavirusof the family Orthomyxoviridae. The genome of ISAVconsists of eight negative-sense single-stranded RNAs. Each segment contains one or more open

reading frames (ORFs) flanked by 5 and 3 untranslated regions (Sandvik et al., 2000; Clouthier et al.,


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2002; McBeath et al., 2006) and together they encode 10 or 11 proteins (Mjaaland et al., 1997;Rimstad and Mjaaland, 2002; Kibenge et al., 2007a). Whereas segments 1 to 6 encode one protein

each, segment 7 encodes two or three proteins and segment 8 encodes two proteins. Functionalcharacterisation of the two viral surface proteins, the HE protein coded by segment 6 and the F protein

coded by segment 5 has disclosed that ISAV possess the major functional characteristics of the

influenza virus family including receptor-binding, fusion and receptor-destroying activities (Falk et al.,1997, 2004; Krossy et al., 2001; Rimstad et al., 2001; Aspehaug et al., 2005). The sequence ofsegments 1, 2 and 4 have been identified to encode the RNA polymerases PB2, PB1 and PA,respectively (Krossy et al., 1999; Clouthier et al., 2002; Snow et al., 2003a) whereas segment 3

encodes a nucleoprotein (Ritchie et al., 2001; Aspehaug et al., 2004). The unspliced mRNA ofsegment 7 encodes a non-structural protein and ORF2 of segment 8 encodes an RNA-bindingstructural protein. Both proteins have interferon antagonistic properties (Biering et al., 2002; McBeathet al., 2006; Garca-Rosado et al., 2008). The spliced mRNA of segment 7 encodes a putative nuclearexport protein (Kibenge et al., 2007b) and ORF1 of segment 8 encodes the matrix protein (Biering etal., 2002; Falk et al., 2004).

3.2. Virulence of ISAVISAV isolates vary in virulence, as observed by differences in disease development and clinical signsin field outbreaks, as well as in experimental trials. In experimental infections, induced mortality fordifferent ISAV isolates varied from 0 % to 47 % in the injected fish and from 3 % to 75 % in thecohabitant fish (Mjaaland et al., 2005).

Only a few potential virulence markers have been identified in the ISAV genome. The functional roleof these markers, if any, has not yet been determined. The HE gene includes an HPR of 35 aminoacids close to the transmembrane region (Devold et al., 2001; Kibenge, Kibenge, et al., 2001; Krossyet al., 2001; Rimstad et al., 2001; Mjaaland, Hungnes, et al., 2002; Falk et al., 2004). Approximately

30 different HPR variants have been identified in Europe, North America and Chile (Nylund et al.,2006; Kibenge et al., 2009; Christiansen et al., 2011). The HPR variants can be explained as variousdeletions (Mjaaland, Hungnes, et al., 2002) from a putative ancestral sequence (HPR0) first identifiedin a wild salmon in Scotland (Cunningham et al., 2002). Whereas all ISAV isolates from ISA diseaseoutbreaks have deletions in the HPR region with respect to the HPR0, the HPR0 subtype has not been

associated with clinical or pathological signs of ISA disease (Cunningham et al., 2002; Cook-Verslootet al., 2004; Nylund et al., 2006; McBeath et al., 2009; Vike et al., 2009; Christiansen et al., 2011).

This strongly suggests that the HPR region is an important virulence marker of ISAV (Mjaaland et al.,2005).

In addition, all virulent strains of ISAV examined so far have the Q266 L266 amino acid substitutionor short amino acid insertions immediately upstream or downstream of the putative arginine cleavagesite R267 in the F protein (Devold et al., 2006; Markussen et al., 2008; Cottet et al., 2011).

However, pathogenic ISAV isolates with identical HPR deletions and mutations around the putativecleavage site R267 of the F gene vary in virulence (Mjaaland et al., 2005; Markussen et al., 2008)suggesting that other as yet unknown genetic changes have an influence on virulence. This issupported by findings in influenza viruses where specific mutations in the PB1 (Conenello et al.,2007) and PB2 (Shinya et al., 2004) genes have been linked to virulence.

3.3. Phylogenetics and evolutionary dynamics of ISAVBased on phylogenetic analysis of segment 2 and segment 8, ISAV isolates have been divided into twomajor genogroups: the North American (NA) genogroup including isolates from Canada and the USA,

and the European (EU) genogroup consisting of ISAV isolates from Norway and Scotland (Blake et al.1999; Krossy et al. 2001). Based on genetic analysis of only the extracellular region of the HE gene

on segment 6, the EU isolates have been further divided into four subgroups (with HPR0 beingrepresented in all subgroups) (Nylund et al., 2003, 2006; Devold et al., 2006; Lyngstad et al., 2008;


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McBeath et al., 2009; Vike et al., 2009; Christiansen et al., 2011). It should be noted that thissubgrouping may not be consistent across different segments (Plarre et al., 2012).

Phylogenetic analysis of Faroese HPR0 ISAV variants identified a lineage with close geneticsimilarity to HPR ISAV associated with the Faroese ISA disease epidemic (Christiansen et al.,

2011). Lyngstad et al (2012) showed that genetic and geographic distances between pairs of HPR0-ISAV sequences are positively correlated, suggesting that the population of HPR0 ISAV isgeographically structured. They also showed that virulent ISAV HE gene sequences from isolated ISA

outbreaks were significantly closer in geographical distance to the genetically closest HPR0 ISAV HEgene sequences than would be expected by chance. This association between genetic and geographical

distance supports the hypothesis that HPR ISAVmay evolve from HPR0 ISAV (Cunningham et al.,2002; Mjaaland, Hungnes, et al., 2002; Nylund et al., 2003).

Several hypotheses have been put forward to explain the origin of HPR and its relationship withHPR0. The original hypothesis was that the virulent HPRISAV is derived from non-virulent HPR0ISAV by deletions in the HPR of the HE molecule (Cunningham et al., 2002; Mjaaland, Hungnes, et

al., 2002).

A postulated insertion hypothesis whereby virulent HPR ISAV undergoes mutations involving

insertions into the HPR, resulting in HPR0 ISAV and thus attenuation (Kibenge et al., 2012), is notconsistent with these findings and does not explain the presence of geographical variants of HPR0.

A third hypothesis brought forward is that HPR0 is the consensus sequence of all HPR sequences

during virus replication (Kibenge et al., 2012). If this hypothesis is correct, fish should be HPR0 ISAVpositive while being infected by a range of virulent HPR ISAV (HPR cloud), of which the

combined HPR consensus sequence would be HPR0. Selection by farming would then be on a varietyof HPRISAV mutants rather than on individual HPR0 ISAV sequences.

Although quasispecies are a common feature of RNA virus infections, i.e. a mixed population of

mutants rather than clonally expanded virus particles, there is no experimental support of an HPRcloud in all ISAV infected fish. Nucleotide sequences from fish found to be HPR0 positive have not

reported the presence of a variety of HPR sequences (Christiansen et al., 2011, Lyngstad 2012).Lyngstad et al (2012) found a geographical relationship between HPR0 and HPR in the nucleotidesequences of the HE gene (excluding the HPR region) (Lyngstad et al., 2012). More recently, the useof new generation sequencing/pyrosequencing of HPR0-positive fish and of ISA-diseased fishdemonstrated the presence of a very small fraction of HPR in HPR0 positive fishand an even smaller

fraction of HPR0 ISAV in diseased fish having one dominant HPR form, thus rejecting the thirdhypothesis (Markussen et al., 2012). The original deletion hypothesis has, on the other hand, beensupported by the findings from sequencing of viral RNA from ISA outbreaks.

To gain further insights into the process of virulence evolution, a quantitative assessment of the

different evolutionary forces for ISA would be useful, in particular the relative contribution ofmutation and reassortment processes. The evolutionary rate of different ISAV segments has beenestimated to be about an order of magnitude lower than that of influenza A viruses (Plarre et al.,2012), but for the HE gene, the rate estimate was also lower than that obtained for a different ISAVdataset (Castro-Nallar et al., 2011). This implies that the tempo and mode of ISAV evolution mayneed further investigation. Although transitional viruses having only the relevant virulence mutationsin either the HE or the F gene have not been identified, reassortment may also be important ingenerating virulent combinations of these segments. Reassortment appears to occur relativelyfrequently for ISA (Plarre et al., 2012), but more formal assessments (e.g., comparisons withinfluenza) are required to determine how pervasive the reassortment is. Finally, the fitness aspects ofthe virulence mutations in the HE and F gene segments remain to be determined, such as thesynergistic or compensatory nature of these mutations, or the preferred evolutionary trajectory towards

virulence.


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4. Relevant risk factors for emergence of HPR (ToR 2b)There is an evolutionary relationship between virulent and low-virulence forms of ISAV, whereby

HPR0 possibly through several steps mutates into a virulent form of the ISAV. However, nopredisposing risk factors have been demonstrated or suggested to drive or increase such anevolutionary process. Hence, there is a lack of knowledge on specific risk factors for HPR

emergence from HPR0. The available evidence on transmission within populations and spreadbetween populations and control thereof is almost entirely based on studies of HPR (ISAV);

however, the observations from the Faroe Islands and Norway have provided some insight into thedynamics of HPR0.

4.1. ISAV transmission and spreadHorizontal transmission is the major pathway for spread of ISA and vertical transmission is notconsidered to play a significant role (Rimstad et al., 2011b). In one study proximity to other ISA-infected holdings and management practices that increased exposure to foreign biological materialwere found to be major risk factors (Vgsholm et al., 1994). The findings indicated that ISA wasspread by infected live salmon or infected biological material, i.e., animal waste or discharge from

normal operations and slaughter.

The knowledge gaps concerning host (Atlantic salmon) susceptibility should be acknowledged. As theISAV (HPR) is found in other species without clinical disease being observed, at least a speciesdifference in susceptibility among salmonid fish appears important. Furthermore, genetic differencesin disease susceptibility following infection with HPR ISAV have been described within Atlanticsalmon (Odegard et al., 2007; Li et al., 2011).

As ISA as a disease has been found only in farmed Atlantic salmon, conditions under which the fish

are farmed are assumed to facilitate this process and possibly provide a positive selection for deletionmutants. This hypothesis fits well with subsequent findings that HPR0 ISAV is widespread in healthy,farmed Atlantic salmon and that it has also been detected in wild Atlantic salmon (Christiansen et al.,

2011).

Strategies for controlling ISA disease have differed between countries, but there is strong evidence

that the disease can be controlled to limit the number of affected farms by the application ofbiosecurity measures that include movement restrictions as well as depopulation and fallowing ofinfected farms (Thorud and Hstein, 2003). It is also possible to eradicate ISA over different scales ofgeographical area in an economically advantageous way by the application and maintenance of goodbiosecurity practice by industry, together with Government regulation (Stagg, 2003; Murray et al.,2010; Christiansen et al., 2011).

The study from the Faroes suggests that HPR0 ISAV is introduced from an endemic source (reservoir)

during the marine phase of the salmon life cycle and that HPR0 ISAV is capable of rapid horizontal

spread through local populations (Christiansen et al., 2011).

Generic control measures such as segregation of generations, caution regarding contact points (water,equipment), sanitary handling of dead fish, cleaning and disinfection, and synchronous fallowing havehad a good effect in terms of the prevention and control of the disease in the Faroe Islands.

A geographically structured pattern is coherent with the hypothesis of an on-going local HPR0 ISAVpropagation in the local biotope (Lyngstad et al., 2012). The presence of HPR0 at various stages in theproduction cycle opens up possibilities for a potential transfer between generations during thestripping/fertilisation phase due to breaks in the biosecurity system and, as such, HPR0 may also becarried by smolts to marine sites (Nylund et al., 2003, 2006).


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4.2. HPRdelta emergence from endemic HPR0 infectionFish susceptibility to virulent viruses may be modulated by acquired immunity. HE is a major antigen

of ISAV and contributes to stimulating a protective immune response (see Section 2.4). Previousinfections with ISAV, like vaccination with attenuated viruses, may induce a level of protection

against subsequent infections with the same or other ISAV variants. The possibility that a subclinical

infection with HPR0 might prevent or mitigate subsequent infections with virulent variants has notbeen investigated.

Industrialised farming of salmon is exposed to a range of environmental challenges, such as highdensity, low physical activity, water with periodically suboptimal oxygen levels and changing

temperatures, intensive feeding and growth rate, and handling. Such exposures may be regarded aschronic or temporary stress inducers, harming their defence mechanisms and aggravating diseaseoutbreak dynamics, as well as potentially influencing the evolution and replication ability of theinfectious agents themselves. Moreover, high-density farming increases the number of possibleinfectious contacts whereby an infection and disease in a single salmon can become a communicabledisease in an industrialised high-density setting.

CONCLUSIONS AND RECOMMENDATIONS

GENERAL CONCLUSIONS

Atlantic salmon is the only species in which the disease ISA has been observed naturally.

ISAV can be genetically differentiated on the basis of the sequence of an HPR of genomic segment 6which encodes the HE protein.

Initial reports of findings of ISAV before 2002 did not distinguish between HPR0 and HPR, makinginterpretation of older findings more difficult in the light of current knowledge.

A single observation of HPR0 ISAV in wild Atlantic salmon indicates that the virus may exist outsidethe farmed salmon population.

TOR1THE CAPABILITY OF HPR0ISAVTO CAUSE CLINICAL DISEASE.

HPR0 ISAV does not cause clinical disease in Atlantic salmon.

It is known that HPR0 ISAV causes a transient subclinical infection and replicates mainly in gills.

There is currently no evidence indicating that HPR0 ISAV leads to natural infection and replication infish species other than Atlantic salmon.

TOR 2 THE RISK OF HPR-DELETED ISA EMERGING FROM HPR0 ISA AND, IF RELEVANT,

INDICATING FACTORS FOR SUCH AN EMERGENCE.

2a Risk of HPR emerging from HPR0

All virulent ISAV have deletions in the HPR region of the HE gene and they have either an insertionor the Q266L mutation in the F gene. Transitional viruses having only one of the HE or F mutationshave not been found.

The most plausible hypothesis is that virulent ISAV (HPR) is derived from HPR0 ISAV.

The close association between genetic relatedness and the spatio-temporal distance of virus strains insolitary outbreaks further supports the hypothesis that virulent ISAV evolve from HPR0-ISAV.

Epidemiological and historical data from solitary disease outbreaks indicate that the risk of emergenceof virulent ISAV is low but not negligible.


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2b Risk factors

There is a lack of knowledge on risk factors for HPR emergence from HPR0.

The risk of emergence of HPR ISAV and subsequent development of disease can be expected to berelated to the overall replication rate of HPR0 ISAV and the presence of susceptible hosts Any factorthat affects replication or host susceptibility would therefore also influence the risk of emergence ofHPR ISAV.

RECOMMENDATION

Baseline monitoring is needed to estimate the prevalence of ISAV HPR0 in farmed Atlantic salmonand wild fish in proximity to farming facilities.

RECOMMENDATIONS FOR RESEARCH

Research should be done to address the drivers for transition from HPR0 to HPR and factorsaffecting host susceptibility and thereby emergence of clinical disease

To gain further insights into the process of virulence evolution, a quantitative assessment of thedifferent evolutionary forces for ISA would be useful, in particular the relative contribution ofmutation and reassortment processes.

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APPENDIX

A. DIAGNOSIS OF ISAAs ISA was described and surveyed for approximately eight years before the causative agent was

identified and described, the diagnosis of the disease was traditionally based on clinical andpathological signs, combined with clinical chemistry, as haematocrit values below 10 in Atlanticsalmon should lead to suspicion of ISA. A number of methods for detection of the virus have since

been developed and have contributed to increasing the sensitivity and specificity of ISA diagnosis.However, the diagnosis of confirmed ISA is still based on a combination of clinical signs and specificdetection of the causative agent ISAV.

The HPR0 variant is not associated with clinical ISA or any ISA-associated pathological changes inany tissue. HPR0 has been detected from fish with gill lesions, but so far no causal association has

been shown or suggested. Findings of HPR0 in apparently healthy fish groups, and often those withlow virus loads (high cycle threshold (Ct) values), support the hypothesis that HPR0 is of lowvirulence (Cunningham et al., 2002; Mjaaland et al., 2002; Markussen et al., 2008; Kibenge et al.,

2009, Lyngstad et al 2011).

The ISA-typical gross macroscopic changes are present late in disease development. At necropsy,

haemorrhage may be found in the liver, kidney, gut and gills. The spleen is often swollen and dark(Mjaaland et al., 1997; Devold et al., 2000; Mikalsen et al., 2001; Snow et al., 2003b). Thehaemorrhagic lesions can be absent or rare in the initial stages of ISA. In the more slowly developing,chronic forms of ISA, clinical signs and pathological changes may be more subtle.

When opening the fish for gross pathological examination the changes can vary from almost none to

severe. No signs are pathognomonic but anaemia is always present, with haematocrit values of < 10 %at the end stage (normal values for non-infected fish > 30 %). The following signs are, however, rather

consistent with ISA:

oedema and yellowish fluid in the abdominal cavity

petechial haemorrhages in the viscera and peritoneum

focal or diffusely dark liver (liver colour is given a number from 1 to 5 according to livercolour card scale used at necropsy in the field)

swollen, dark-red spleen with rounded margins

swollen, dark-red kidney with blood and liquid effusing from cut surfaces

pinpoint haemorrhages of especially the dorsal skeletal muscle.

ISA virus can be detected by a range of direct and indirect immunochemical and molecular methods.

According to the World Organisation for Animal Health (OIE), the following criteria should be metfor confirmation of ISA: mortality, clinical signs and pathological changes consistent with ISA anddetection of ISAV in tissue preparations by means of specific antibodies against ISAV, in addition to

either isolation and identification of ISAV in cell culture or detection of ISAV by RT-PCR.

According to OIE the definition of confirmed ISAV HPR infection requires two criteria to be met:

isolation and identification of ISAV in cell culture from at least two independent samples

(targeted or routine) from any fish on the farm tested


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isolation and identification of ISAV in cell culture from at least one sample from any fish on

the farm with corroborating evidence of ISAV in tissue preparations using either RT-PCR orimmunofluorescent antibody test (IFAT)/immunohistochemistry (IHC).

Similarly, the confirmation of infection with HPR0 ISAV requires:

an absence of clinical signs consistent with ISA disease or mortality (= apparently healthy

fish)

detection of ISAV by RT-PCR followed by independent amplification and sequencing of the

HPR region of segment 6 to confirm the presence of HPR0 only.

Propagation of ISAV in cell lines

The HPR ISAV variants can be propagated in various cell cultures. The fi rst cell line supporting

replication of ISAV was SHK-1, established from a culture of Atlantic salmon head kidney cells.Cytopathic effect (CPE) was observed 1214 days after inoculation (Dannevig et al., 1995). Morerecently Devold et al. (2000) and Wergeland and Jakobsen (2001) reported the isolation of an Atlantic

salmon head kidney cell line (ASK) and TO cell line, respectively, that supported replication of HPRISAV showing CPE in only seven to eight days. Other cell lines have also been used to propagate

HPR ISAV; however, they all display low yields of virus and poorly defined CPE compared withSHK-1, TO and ASK.

Only ISAV HRP isolates have hitherto been able to multiply in cell cultures, and even among theseisolates CPE is not always obtained. When cultivating, cell cultures with no CPE should always betested in an haemadsorption test using salmonid erythrocytes, by IFAT or by RT-PCR, as virus

replication regularly occurs without development of apparent CPE. ISAV in cell cultures is identifiedby antibody-based antigen detection methods such as IFAT or by molecular techniques such as RT-

PCR or real time RT-PCR.

Although several laboratories have tried to propagate and isolate HPR0 ISAV in conventional ISAVpermissive cell lines they have so far been non-culturable with no cytopathic effect (Nylund et al.,2006). Despite the unsuccessful attempts to cultivate ISA HPR0, it was recently demonstrated byChristiansen et al. (2011) that ISA HPR0 could in fact multiply in ASK cells as they demonstrated that

both ORF1 and the cellular splicing dependent ORF1/2 and possibly the ORF1/3 of segment 7 couldbe detected in HPR0-infected ASK cells as a means of proving cellular translation. Whether the

multiplication resulted in fully developed virions or not remains to be shown.

Direct detection methods

ISAV can be detected directly in fish tissue material by IFAT on smears of kidney imprints usingmonoclonal antibodies, or by IHC, in which the strongest staining usually is observed in endothelial

cells of heart and kidney, or, as is most widely used, by RT-PCR on tissue material.

ISAV HPR0 infection is found on the gills, and this organ has to be collected if a survey targetingISAV HPR0 is conducted. ISA HPR0 is, however, occasionally detected also in internal organs. As nospecific immunochemical or PCR-based methods have been developed yet, ISAV HPR0 can so far beconfirmed only through sequencing the ISAV-positive PCR-products of segment 6.

Detection of ISAV (HPR) by RT-PCR (interpretation for surveillance and reservoirs)

ISAV HPR is identified by molecular methods and often also genotyped and characterised bysubsequent sequencing. The primers described in the OIE manual for RT-PCR and real-time RT-PCRwill detect both HPR0 and HPR HPR ISAV, and the methods may be used for detection of ISAV in

tissue samples or in samples from cell cultures. Since Mjaaland et al. (1997) reported the first RT-PCRfor ISAV, which targeted genomic segment 8, several methods have been reported.


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EFSA Journal 2012;10(11):2971 21

The primer sets derived from genomic segment 8 (ILA1/ILA2) have been used by several laboratoriesand have been found suitable for detection of ISAV during disease outbreaks and in carrier fish (ILA2

reverse primer does not, however, match isolates from North America). The segment 6 primers may beuseful for verification of PCR results based on segment 8 primers as an alternative to sequencing the

PCR product. With the widespread occurrence of HPR0 variants in mind, for any positive PCR results

based on the segment 8 primers, it is essential to follow up by sequencing the HPR of segment 6 inorder to determine the ISAV HPR variant present (HPR or HPR0 or both). For this purpose theprimers described by Kibenge et al. (2009) are adequate.

Table 1: Primers targeting segment 6 and 8, respectively, in conventional RT-PCR

RT-PCR: primer sequences Named Genomic

segment

Product

size

Reference

5 -GGC-TAT-CTA-CCA-TGA-ACG-AAT-C-3 5 -GCC-AAG-TGT-AAG-TAG-CAC-TCC-3

ILA1ILA2

8 155 Mjaaland

et al., 2002b

5 -GGA-ATC-TAC-AAG-GTC-TGC-ATT-G-3 5 -CTT-CAA-AGG-TGT-CTG-ACA-CGT-A-3

Seg6USeg6L

6 130 Designed byOIE Ref. Lab.

5 -GCC-CAG-ACA-TTG-ACT-GGA-GTA-G-3

5 -AGA-CAG-GTT-CGA-TGG-TGG-AA-3

ISAV HPR Fwd

ISAV HPR Rev

6 319391 Kibenge et

al., 2009

The use of real-time RT-PCR has several advantages, when compared with conventional RT-PCR, interms of its higher analytical specificity and often also sensitivity, especially when including a

sequence-specific probe. This method is also more rapid, the risk of contamination is reduced and atthe same time it is possible to estimate the relative amount of viral RNA in the sample, but none of thehitherto published methods are yet able to discriminate between ISAV HPR0 and HPR

Snow et al. 2006) developed a real-time RT-PCR with primer and probe sequences targeting segment

7 and 8, respectively, that have been used for screening for ISAV. Both primers and probes targetconserved regions and ensure detection of all documented ISAV strains, and a positive real-time RT-PCR will thus demand a subsequent sequencing of segment 6 after a conventional RT-PCR.

Table 2: Primers targeting segment 7 and 8, respectively, in real-time RT-PCR

Real-time RT-PCR: primer andprobe sequences Named Genomicsegment

Reference

5 -CAGGGTTGTATCCATGGTTGAAATG-3 5 -GTCCAGCCCTAAGCTCAACTC-3 5 -6FAM-CTCTCTCATTGTGATCCC-MGBNFQ-3

Forward primerReverse primerTaqmanprobe

7 Snow et al.,2006

5 -CTACACAGCAGGATGCAGATGT-3 5 -CAGGATGCCGGAAGTCGAT-3 5 -6FAM-CATCGTCGCTGCAGTTC-MGBNFQ-3

Forward primerReverse primerTaqmanprobe

8 Snow et al.,2006


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GLOSSARY AND ABBREVIATIONS

F Fusion protein

HE Hemagglutinin-Esterase

HPR Highly Polymorphic Region

HPR0 Low-virulence ISAV having a full-length HPR

HPR Virulent ISAV having a deletion in HPR

ISA Infectious Salmon Anaemia

ISAV Infectious Salmon Anaemia Virus

COMMON AND LATIN NAMES OF SPECIES MENTIONED IN THE TEXT.

Latin name Common name

Alosa pseudoharengus Alewife

Oncorhynchus kisutch Coho salmon

Gadus morhua Atlantic cod

Mytilus edulis Blue mussel

Oncorhynchus mykiss Rainbow trout

Pecten maximus Scallop

Pollachius pollachius Pollock

Pollachius virens Saithe

Salmo salar Atlantic salmon

Salmo trutta Brown trout; Sea trout

ISA_V paper

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